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Abstract

Ferns of the Swat region represent a rich component of Pakistan’s flora, yet they remain unexplored at molecular and phylogenetic level. Specimens representing several fern species were collected from four areas of Swat Valley located in the Hindukush-Himalayan mountain range of Pakistan. The specimens, initially identified based on morphology, were subjected for the first time to molecular and phylogenetic analyses for DNA barcoding using sequences of the rbcLa marker (a segment of rbcL gene). This chloroplast marker was successfully amplified and sequenced using Sanger sequencing method. Genetic identification or DNA barcoding at the species level was conducted using three DNA based datasets approaches: BLASTn-based sequence similarity searches, genetic distance, and maximum likelihood (ML) phylogenetic reconstruction. All three methods yielded congruent and reliably authenticated results for genetic diversity and DNA barcoding of sampled fern taxa. The rbcLa barcode exhibited strong discriminatory power and clearly resolved species within their respective clades, which was statistically supported by the Mann-Whitney U test (Z = −7.615, p<0.001) and ROC analysis (AUC = 0.991). This study authenticated three species of Adiantum and two species of Pteris through rbcLa-based molecular identification. It also provides molecular evidence for taxonomic status of Onychium lucidum (D. Don) Spreng and O. cryptogrammoides Christ, which united in phylogenetic tree suggesting that O. contiguum should be treated as O. cryptogrammoides. Also, the presence of Aleuritopteris acrostica (Balb.) Windham & Schuettp. [Hemionitis acrostica (Balb.) Mosyakin] and A. nitidula (Hook.) Windham & Schuettp. [H. nitidula (Hook.) Christenh] has been uncertain in Pakistan, our results provided the first DNA-based confirmation of their presence in the country and in fern flora of Swat. Overall, our findings highlighted the effectiveness of rbcLa for authentication of species of Pteridaceae ferns and provided the first molecular insight into fern flora of the Swat Valley lacking DNA-based systematic documentation, while also suggesting that incorporation of additional plastid or nuclear markers may further improve species level resolution in future studies

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